Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody

Abstract

Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed "TKD," comprising amino acids 450-461 (aa(450-461)) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70(+) mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.

Fig. 1.

Representative flow cytometric histograms of membrane Hsp70 expression on human and mouse tumor cell lines. (A) Human CX2 (colon), MDA436 (breast), MCF-7 (breast), Malme, Mel Ei, Mel Ho, Parl, A375, Sk Mel29 (all malignant melanomas), A549 (lung) carcinoma, (B) primary and immortalized endothelial cells, and (C) mouse CT26 (colon), 1048 (pancreas), A20 (B lymphoma), B16F0 (low-malignant melanoma), and (B) B16F10 (high-malignant, metastatic melanoma), tumor cell lines were analyzed by flow cytometry using either cmHsp70.1-FITC (Upper) or SPA810-FITC (Lower) mAb. Results are expressed as log green fluorescence intensity vs. relative cell numbers. The IgG1 isotype-matched control is indicated in white and membrane Hsp70 staining in gray histograms. The whole staining procedure was performed at 4 °C and only viable, 7-AAD⁻ cells were gated upon and analyzed. (B) Comparison of the membrane Hsp70 expression in isogenic human and mouse cells. Human primary endothelial cells (ECs, Top and Middle) were compared with their corresponding immortalized partner cell lines EA.hy926, a fusion product of human umbilical vein ECs (HUVEC) with A549 lung carcinoma cells, and HMEC, which was derived by a transfection of primary ECs with SV40 large T antigen, were stained with cmHsp70.1 mAb, as described above. Furthermore, the low-malignant mouse melanoma cell line B16F0 (Left) was compared with the high-malignant, metastatic tumor cell line B16F10. The IgG1 isotype-matched control is indicated in white and membrane Hsp70 staining using cmHsp70.1 mAb in gray histograms. (D) The flow cytometric analysis of CT26 and 1048 tumor cell lines using cmHsp70.1-FITC mAb was repeated six times. The differences in membrane Hsp70 positivity in CT26 and 1048 tumor cell lines was significant (*P < 0.05).

Fig. 2.

Comparative analysis of ADCC using different IgG1 mAbs and cmHsp70.1 Fab fragment. (A) In vitro ADCC of membrane Hsp70⁺ mouse CT26 colon (60%, filled squares) and membrane Hsp70⁻ 1048 pancreatic carcinoma cells (filled diamonds), using 50 μg/mL Hsp70.1 mAb and unstimulated mouse spleen cells at E:T ratios ranging from 50:1 to 6.25:1. (B) In comparison with cmHsp70.1 mAb (filled squares), no significant ADCC was induced in mouse CT26 colon carcinoma cells (61% cmHsp70.1⁺) using the nonbinding IgG1 mAb SPA810 (filled circles) or cmHsp70.1 Fab fragment (filled triangles). (C) The IgG1 Ox7.11 mAb, which detects the theta antigen on 56% of the BW mouse tumor cells, does not induce ADCC in BW mouse tumor cells (filled triangles). Specific ADCC was measured using 50 μg/mL antibody or Fab fragment, respectively; unstimulated mouse spleen cells at E:T ratios ranging from 50:1 to 6.25:1 were used as effector cells. Specific lysis mediated by the direct cytotoxic effect of NK cells in the absence of cmHsp70.1 mAb was subtracted. The phenotypes of the effector cells are summarized in Table 2. Data are means ± SE of at least three independent experiments (**P < 0.01; *P < 0.05). (D) Comparative analysis of the capacity of unstimulated (ctrl, open circles; ctrl+Ab, filled diamonds) and TKD (2 μg/mL) plus IL-2 (100 IU/mL) preactivated (TKD/IL-2, open triangles; TKD/IL-2+Ab, closed squares) mouse spleen cells to kill CT26 carcinoma cells. The ADCC experiment was performed either in the absence (open symbols) and presence (+Ab; closed symbols) of 50 μg/mL cmHsp70.1 mAb. Lysis is mediated by ADCC in the presence of cmHsp70.1 mAb and by a direct cytotoxic effect of mouse NK cells in the absence of mAb, at E:T ratios ranging from 25:1 to 0.38:1. Data are means ± SE of at least three independent experiments. Lysis of activated effector cells in the absence and presence of cmHsp70.1 mAb was significantly different (*P < 0.05, all E:T ratios). ADCC was calculated using the formula: percent of specific lysis = (experimental release − spontaneous release)/(maxiumum release − spontaneous release) × 100.

Fig. 3.

Reduction in tumor weight and the delay of CT26 tumor growth in BALB/c mice after one to three injections of cmHsp70.1 mAb is associated with an infiltration of immunocompetent effector cells. (A) Two and three consecutive injections of cmHsp70.1 mAb (intravenously) result in a significant reduction in tumor weight (*P < 0.05). The cmHsp70.1 mAb (20 μg per injection) was injected intravenously on days 3, 5, and 7 following intraperitoneal injection of 2.5 × 10⁴ CT26 tumor cells. Mice were killed on day 14 and tumor weights were determined. Data are means of six to nine animals (*P < 0.05). (B) Representative photomicrographs of CT26 tumor sections after one (1 × Ab; Left) and three consecutive injections of cmHsp70.1 mAb (3 × Ab; Right) on days 3, 5, and 7 (20 μg per injection). Infiltration of NK cells (CD56; Top), monocytes (T4/80; Middle) and granulocytes (Ly6G/Ly6C; Bottom) was determined on consecutive sections of CT26 tumors derived from mice on day 14. Semiquantitative data are summarized in Table 2. (Scale bar, 100 μm.) (C) Three (filled square) but not one (filled triangle) injections of cmHsp70.1 mAb (i.v.) result in a significant growth delay of subcutaneously injected CT26 tumors (*P < 0.05). The cmHsp70.1 mAb (20 μg per injection) was injected intravenously on days 4, 7, and 10 following subcutaneous injection of 1 × 10⁶ CT26 tumor cells. Tumor weight was measured in each mouse every second day after the last antibody injection (*P < 0.05 for all time points from day 10 onwards). (D) Control mice (open circles) and mice that were injected only once with mAb cmHsp70.1 (filled circles, day 5) became moribund from day 18 onwards, whereas mice that were injected three times (filled squares, day 4, 7, 10) with cmHsp70.1 mAb showed a significant increase in overall survival (*P < 0.05). Each datapoint represents measurements of six to nine mice.

Fig. 4.

(A) Kaplan-Meyer curves of overall survival of mice treated with an isotype-matched control antibody or cmHsp70.1 mAb on days 3, 5, and 7 after intraperitoneal injection of 2.5 × 10⁴ CT26 tumor cells (20 μg per injection). The overall survival of mice (3 × Ab cmHsp70.1, filled squares; n = 24) treated with cmHsp70.1 mAb was significantly higher than that of animals (ctrl, open circles; n = 14) that received the IgG1 isotype-matched control antibody (P < 0.0001). (B) In contrast, the cmHsp70.1-mAb treatment (3 × Ab cmHsp70.1, filled squares) had no significant effect on the survival of mice bearing membrane Hsp70⁻ A20 lymphomas (n = 12) compared with mice receiving the IgG1 isotype-matched control antibody (ctrl, open circles; n = 4, P = 0.310).

Fig. 5.

(A) Coinjection of an excess of the Hsp70 peptide TKD with the cmHsp70.1 mAb completely inhibits the significant antitumoral effect of the latter (*P < 0.02). The cmHsp70.1 mAb (20 μg per injection) was coinjected intravenously on days 3, 5, and 7 together with 50 μg TKD following intraperitoneal injection of 2.5 × 10⁴ CT26 tumor cells; media, n = 21; Ab, n = 22; Ab+TKD, n = 21. Mice were killed on day 14 and tumor weights were determined. (B) Binding of cmHsp70.1-FITC mAb to CT26 tumor cells was inhibited by the coincubation with an excess of TKD peptide. As a control, the scrambled NGL peptide was used. Tumor cells were coincubated either with cmHsp70.1-FITC mAb (5 μg/mL; white histogram) or with cmHsp70.1-FITC mAb (5 μg/mL) plus TKD (gray histogram; Left) or NGL peptide (gray histogram; Right) at concentrations of 12.5 and 25 μg/mL, respectively. The data illustrate one representative experiment out of three independent experiments, all of which show similar results. (C) Binding of cmHsp70.1-FITC to CT26 tumor cells was inhibited significantly (*P < 0.05) by the coincubation with the C-terminal substrate-binding domain of Hsp70 in a concentration dependent manner (10 and 50 μg/mL).